Cyclic di-GMP Regulates the Type III Secretion System and Virulence in Bordetella bronchiseptica.

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.

Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion.

Rhomboids are intramembrane serine proteases highly conserved in the three domains of life. Their key roles in eukaryotes are well understood but their contribution to bacterial physiology is still poorly characterized. Here we demonstrate that Brucella abortus, the etiological agent of the zoonosis called brucellosis, encodes an active rhomboid protease capable of cleaving model heterologous substrates like Drosophila melanogaster Gurken and Providencia stuartii TatA. To address the impact of rhomboid deletion on B. abortus physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. About 50% of the B. abortus predicted proteome was identified by quantitative proteomics under two experimental conditions and 108 differentially represented proteins were detected. Membrane associated proteins that showed variations in concentration in the mutant were considered as potential rhomboid targets. This class included nitric oxide reductase subunit C NorC (Q2YJT6) and periplasmic protein LptC involved in LPS transport to the outer membrane (Q2YP16). Differences in secretory proteins were also addressed. Differentially represented proteins included a putative lytic murein transglycosylase (Q2YIT4), nitrous-oxide reductase NosZ (Q2YJW2) and high oxygen affinity Cbb3-type cytochrome c oxidase subunit (Q2YM85). Deletion of rhomboid had no obvious effect in B. abortus virulence. However, rhomboid overexpression had a negative impact on growth under static conditions, suggesting an effect on denitrification enzymes and/or high oxygen affinity cytochrome c oxidase required for growth in low oxygen tension conditions.

Quantitative proteomic analysis reveals altered enzyme expression profile in Zea mays roots during the early stages of colonization by Herbaspirillum seropedicae.

The use of plant growth-promoting bacteria as agricultural inoculants of plants should be encouraged because of their prominent role in biological nitrogen fixation, the increase of nutrient uptake by roots, abiotic stress mitigation, and disease control. The complex mechanisms underlying the association between plant and beneficial bacteria have been increasingly studied, and proteomic tools can expand our perception regarding the fundamental molecular processes modulated by the interaction. In this study, we investigated the changes in protein expression in maize roots in response to treatment with the endophytic diazotrophic Herbaspirillum seropedicae and the activities of enzymes related to nitrogen metabolism. To identify maize proteins whose expression levels were altered in the presence of bacteria, a label-free quantitative proteomic approach was employed. Using this approach, we identified 123 differentially expressed proteins, of which 34 were upregulated enzymes, in maize roots cultivated with H. seropedicae. The maize root colonization of H. seropedicae modulated the differential expression of enzymes involved in the stress response, such as peroxidases, phenylalanine ammonia-lyase, and glutathione transferase. The differential protein profile obtained in the inoculated roots reflects the effect of colonization on plant growth and development compared with control plants.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Revisiting the stem proteome of Eucalyptus grandis and Eucalyptus globulus: Identification of temperature-induced changes.

Eucalyptus grandis and Eucalyptus globulus are important species for the Brazilian forestry industry. E. grandis plantations are mainly found in tropical regions, yet E. globulus plants are usually cultivated under moderate to low temperature conditions. As temperature seems to be a key factor for the planting of these species, we revisited our previously generated shotgun proteomics dataset to identify the main patterns of proteome regulation induced by thermal stimulus and to pinpoint specific proteins involved in the environmental response. Large-scale analysis has pointed out the different proteomic responses of E. grandis and E. globulus under temperature stimulus, with 296 proteins considered to be differentially regulated in the stems of Eucalyptus spp. grown at different temperatures. A stringent filtering approach was used to identify the most differentially regulated proteins. Through the stringent criteria, 66 proteins were found to be enriched in the plant species. Cultivation of E. globulus plants in low-temperature conditions induced the highest number of differentially regulated proteins. Additionally, metabolic proteins were mostly down-regulated, while stress-related proteins were majorly up-regulated in both species. Finally, the subset of the most differentially regulated proteins comprised new candidates of protein markers of temperature stress.

LED lamps enhance somatic embryo maturation in association with the differential accumulation of proteins in the Carica papaya L. 'Golden' embryogenic callus.

The use of light-emitting diode (LED) lamps has been shown to be a promising approach for improving somatic embryo maturation during somatic embryogenesis. The aim of this work was to study the influence of the light source on somatic embryo differentiation and its relationship with the differential abundance of proteins in the Carica papaya L. 'Golden' embryogenic callus at 14 days of maturation. The white plus medium-blue (WmB) LED and fluorescent lamp treatments produced an average of 82.4 and 47.6 cotyledonary somatic embryos per callus, respectively. A shotgun proteomics analysis revealed 28 upaccumulated and 7 downaccumulated proteins. The proteins upaccumulated in the embryogenic callus matured under the WmB LED lamp compared with that matured under the fluorescent lamp included indole-3-acetic acid-amido synthetase (GH3) and actin-depolymerizing factor 2 (ADF2), which are involved in the regulation of auxin levels by auxin conjugation and transport. Additionally, proteins related to energy production (aconitate, ADH1, GAPCp, PKp and TPI), cell wall remodeling (PG and GLPs), and intracellular trafficking (NUP50A, IST1, small GTPases and H+-PPase) showed significantly higher abundance in the embryogenic callus incubated under the WmB LED lamp than in that incubated under the fluorescent lamp. The results showed that the WmB LED lamp improved somatic embryo maturation in association with the differential accumulation of proteins in the C. papaya 'Golden' embryogenic callus.

Quantitative Proteomic Analysis of the Response of Probiotic Putative Lactococcus lactis NCDO 2118 Strain to Different Oxygen Availability Under Temperature Variation.

Lactococcus lactis is a gram positive facultative anaerobe widely used in the dairy industry and human health. L. lactis subsp. lactis NCDO 2118 is a strain that exhibits anti-inflammatory and immunomodulatory properties. In this study, we applied a label-free shotgun proteomic approach to characterize and quantify the NCDO 2118 proteome in response to variations of temperature and oxygen bioavailability, which constitute the environmental conditions found by this bacterium during its passage through the host gastro-intestinal tract and in other industrial processes. From this proteomic analysis, a total of 1,284 non-redundant proteins of NCDO 2118 were characterized, which correspond to approximately 54% of its predicted proteome. Comparative proteomic analysis identified 149 and 136 proteins in anaerobic (30°C and 37°C) and non-aerated (30°C and 37°C) conditions, respectively. Our label-free proteomic analysis quantified a total of 1,239 proteins amongst which 161 proteins were statistically differentially expressed. Main differences were observed in cellular metabolism, stress response, transcription and proteins associated to cell wall. In addition, we identified six strain-specific proteins of NCDO 2118. Altogether, the results obtained in our study will help to improve the understanding about the factors related to both physiology and adaptive processes of L. lactis NCDO 2118 under changing environmental conditions.

Characterization of extracellular proteins in members of the Paracoccidioides complex.

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) - an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MSE) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes.

Targeting Hodgkin and Reed-Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance.

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol's toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc-Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label-free shotgun 2D-nanoUPLC/MSE to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc-susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large-scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant-pathogen interaction in planta.

Intrauterine Growth Restriction Programs the Hypothalamus of Adult Male Rats: Integrated Analysis of Proteomic and Metabolomic Data.

Programming of hypothalamic functions regulating energy homeostasis may play a role in intrauterine growth restriction (IUGR)-induced adulthood obesity. The present study investigated the effects of IUGR on the hypothalamus proteome and metabolome of adult rats submitted to 50% protein-energy restriction throughout pregnancy. Proteomic and metabolomic analyzes were performed by data independent acquisition mass spectrometry and multiple reaction monitoring, respectively. At age 4 months, the restricted rats showed elevated adiposity, increased leptin and signs of insulin resistance. 1356 proteins were identified and 348 quantified while 127 metabolites were quantified. The restricted hypothalamus showed down-regulation of 36 proteins and 5 metabolites and up-regulation of 21 proteins and 9 metabolites. Integrated pathway analysis of the proteomics and metabolomics data indicated impairment of hypothalamic glucose metabolism, increased flux through the hexosamine pathway, deregulation of TCA cycle and the respiratory chain, and alterations in glutathione metabolism. The data suggest IUGR modulation of energy metabolism and redox homeostasis in the hypothalamus of male adult rats. The present results indicated deleterious consequences of IUGR on hypothalamic pathways involved in pivotal physiological functions. These results provide guidance for future mechanistic studies assessing the role of intrauterine malnutrition in the development of metabolic diseases later in life.

Early downregulation of acute phase proteins after doxorubicin exposition in patients with breast cancer.

Chemotherapy remains the first-choice option for adjuvant therapy in breast cancer. Here, we investigated the impact of the first chemotherapic cycle of doxorubicin on the plasmatic-proteomic profiling of women diagnosed with breast cancer (n = 87). Blood samples were obtained from the same patient before and after doxorubicin infusion (1 h, 60 mg/m(2)) and processed for label-free LC-MS proteomic screening. A total of 80 proteins were downregulated after chemotherapy. In silico analysis revealed that the main biological process enrolled was inflammation and canonical pathways involving acute phase proteins. TNF-α, IL-1ß, IL-12, TGF-ß1, clusterin, and gelsolin were chosen as relevant for further validation. All selected targets presented reduced plasmatic levels after treatment. Our results indicate that doxorubicin downregulated acute phase proteins immediately after its infusion. Since such proteins are cancer promoting, its downregulation could support the effectiveness of doxorubicin along treatment.